RELATIONSHIP BETWEEN DIETARY CARBOHYDRATES AND FATS IN THEIR INFLUENCE ON SERUM LIPID CONCENTRATIONS

Clinical Science (1972) 43,265-214. RELATIONSHIP BETWEEN DIETARY CARBOHYDRATES AND FATS IN THEIR INFLUENCE ON SERUM LIPID CONCENTRATIONS I. MACDONALD Department of Physiology, Guy s Hospital Medical School, London (Received 26 January 1972) SUMMARY 1. Dietontaining 45% joules as fat, 45% joules aarbohydrate and 10% joules aalcium caseinate were given for 5 days to ten men and seven women. The fats used were sunflower seed oil or cream, and the carbohydrates were either glucose and fructose, glucose and raw starch or fructose and raw starch. 2. Sunflower seed oil in the diet resulted, after 5 days, in a significant fall in the concentration of triglyceride, cholesterol and phospholipid in the serum taken from subjects fasted for 12 h irrespective of the carbohydrate mixture of the diet. 3. Although there were changes in serum lipid concentrations in relation to the different mixtures of carbohydrate they were much less than those seen after ingesting sunflower seed oil. 4. Men and women differed in their response to carbohydrates, but this distinction was not seen in response to dietary fats. Key words : dietary fats, dietary carbohydrates, serum lipids. Saturated fat in the diet is frequently associated with a rise in the serum cholesterol concentration whereas polyunsaturated fat in the diet may decrease a raised serum cholesterol concentration (Keys, 1970). Also, dietary carbohydrate can raise the concentration of triglycerides in the serum of fasting subjects (Ahrens, Hirsch, Little, Farquhar & Stein, 1961). Little work, however, has been reported on the interrelationship of fat and carbohydrate in the diet with lipid metabolism. It has been reported that the increase in triglyceride level of fasting serum in response to dietary sucrose can be modified by the type of fat in the diet both in normal men (Macdonald, 1967) and hypertriglyceridaemic patients (Antar, Little, Lucas, Buckley & Csima, 1970). The experiments described in the present paper were done to learn to what extent the type of dietary fat could influence the response of serum lipids in fasting subjects to various dietary carbohydrate mixtures. Correspondence: Professor I. Macdonald, Department of Physiology, Guy s Hospital Medical School. London, S.E.1. 265

I. Macdonald MATERIALS AND METHODS Ten healthy male dental students aged 18-21 years volunteered to subsist on a liquid formula diet composed of carbohydrate (45% of joules), fat (45% of joules) and calcium caseinate protein (10% ofjoule intake). One vitamin capsule (B.P.C.) was taken by each subject on each day during the dietary period, which lasted 5 days. There was an interval of at least 10 days between each diet, and each subject participated in all six experimental diets. The intake of formula diet was approx. 170 J day-' kg body weight-' and was adjusted to keep the body weight constant. As a difference in the triglyceride response to dietary carbohydrate has been observed between men and pre-menopausal women (Macdonald, 1965) it was also decided to give seven young women aged 18-21 years diets similar to those given to the men. The fats used were sunflower seed oil or double cream (48% volume = fat). The carbohydrate mixtures used were either: (a) 40% fructose and 60% raw corn starch, (b) 40% fructose and 60% glucose, or (c) 40% glucose and 60% raw corn starch. The carbohydrates were mixed in this way because it is not possible to use fructose as the sole source of dietary carbohydrate, as it may cause diarrhoea. Samples of serum were obtained after a 12 h fast on the morning the diet started and on the penultimate and final mornings before the subject returned to his free-choice diet. The triglyceride (Lofland, 1964), phospholipid (Krehl, Lopez & Good, 1967) and cholesterol (Block, Jarret & Levine, 1966) estimations were semi-automated. TABLE 1. Weight and 'declared' normal intakes of the men before the experimental period (valuealculated from McCance & Widdowson, 1960) Energy content Subject Weight Protein Fat Carbohydrate Sucrose Alcohol (kg) (g) (9) (g) (9) (g) (cal) (kj) S.M. A.W. D.D. J.W. T.G. M.M. E.H. P.M. M.G. J.T. Mean 84 78 13 75 18 75 72 93 73 68 77 100 90 75 80 75 105 80 90 120 85 90 110 120 100 95 105 155 110 120 135 135 119 235 305 280 300 190 365 365 395 375 345 316 50 25 70 85 5 25 125 150 80 95 71 10 25 55 30 10 10 60 40 5 20 27 2400 2850 2700 2600 2100 3350 3200 3300 3200 2950 2865 10.1 12.0 11.3 10.9 8.8 14.1 13.4 13.9 13-4 12-4 12.0 RESULTS Men The weight of each subject and average dietary intake before the experiment are seen in Table 1. Lipid concentration in serum during fasting. The mean values for each subject obtained during fasting from six serum samples on the day each diet started are seen in Table 2. Due to

TABLE 2. Mean of six values of the pre-diet concentration (mg/loo ml) of triglyceride, cholesterol and phospholipid in serum from fasting subjects a?i Men Women "- c - Subject Triglyceride Cholesterol Phospholipid Subject Triglyceride Cholesterol Phospholipid Weight S.M. A.W. D.D. J.W. T.G. M.M. E.H. P.M. M.G. J.T. Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE 79 103 83 68 83 122 106 213 96 84 4.4 4.2 4.5 6.3 4.2 6.4 10.3 9.9 10.2 7.9 161 222 193 139 183 154 191 199 189 178 7-2 175 10.4 9-1 244 13-9 8.0 193 14.2 6.8 156 9.2 2-7 201 12.9 4.0 152 12.2 5.7 198 14.3 12-1 202 15.4 5-2 181 18.5 10.6 169 15.6 * Subject partook of three experimental diets only. (kg) $ 0.;r J.T. 95 6.0 201 3.1 148 5.5 70 A.S. 107 6.4 208 9.7 160 11.4 50 D.W. 84 6.2 168 3.4 143 4.7 51 4 A.C. 70 5.0 158 4.5 134 5.3 59 J.S.* 102-229 - 166-6 0 % R.B. 109 9.8 221 5.4 155 5.8 61 GI J.R. 70 6.5 198 3.2 142 5.3 68 f k! 3 g t.l %

TABLE 3. Mean percentage change in serum lipid fractions after fasting in men Trig1 yceride Cholesterol Phospholipid Diet n Day 4 Day 5 Day 4 Day 5 Day 4 Day 5 Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE 5 Sunflower seed oil, fructose, starch 10-10 61-12* 3.7-17* 2.0-22* 2.6-33* 3.7-39* 3.2 Cream, fructose, starch 10 +13 7.2 $22 11.5 +8* 2.3 +12* 2.6 +8* 2.3 +12* 2.6 Sunflower seed oil, fructose, glucose 10-23* 4.0-29* 3.8-27* 1.9-30* 2.3-21* 4.1-30* 1.5 Cream, fructose, glucose 10-5 3.2-6 4.8-2 1.2-1 1.7-5 3.4 0 2.9 Sunflower seed oil, glucose, starch 10-24* 2.9-25* 4.1-23* 2.0-28* 1.9-28* 3.0-27* 2.1 Cream,glucose,starch 10 +22* 5.1 +26* 10.3-1 2.3 f3 2.3-16 6.5-12 6.2 * P< 0.025.

Fats, carbohydrates and serum lipids 269 wide individual range of values the changes after 4 and after 5 days on each experimental diet for each lipid component were expressed, for every subject, as a percentage rise or fall from his mean fasting value. The mean values of each of these percentage changes in each lipid fraction for each experiment diet are seen in Tables 3,4 and 5. Triglyceride (Table 3). The triglyceride concentration in the serum taken from subjects fasted for 12 h fell significantly after 5 days on the diet containing sunflower seed oil, whereas on the diet containing cream, glucose and starch there was a significant rise in the fasting triglyceride concentration after days 4 and 5. To assess differences between diets the percentage change in the lipid fraction produced by one diet on day 4 and on day 5 was subtracted from the corresponding value for that individual on comparable days while on another diet. The mean of the ten values for each day, so obtained, was tested using Student s t test for significant difference from zero. + 20 + 12 +4 0-4 - 12-20 Fructose -Glucose d? J Starch -Glucose? r d FIG. 1. Mean percentage difference in serum triglyceride concentrations between diets. S, sunflower seed oil; C, cream. Shaded area, P<O.O25. When this was done for the triglyceride concentration in serum from fasting subjects, it was found that after ingestion of sunflower seed oil, the triglyceride concentration was higher when fructose was in the diet than when glucose was in the diet. This effect was not seen with cream in the diet. Also, with both dietary fats the triglyceride concentration in the serum was higher when starch replaced glucose (Fig. 1). Cholesterol (Table 3). As expected the cholesterol concentration decreased when sunflower seed oil was in the diet. By subtracting the results for each subject on one diet from the results in the same subject on another diet it was found that on all the carbohydrate diets the cholesterol concentration after the sunflower seed oil diet was approx. 30% lower than after the cream diet. The diets with cream contained cholesterol (1-2 mg/ml).

270 I. Macdonald + 16 Fructose - Glucose d sqc Starch - Glucose d sqc + 12 +8 I FIG. 2. Mean percentage difference in serum cholesterol concentrations between diets. S, sunflower seed oil; C, cream. Shaded area, P<0.025. +4 0-4 -8 c4 Fructose - Glucose d I Starch - Glucose d? 0-4 - 12-20 - 28 FIG. 3. Mean percentage difference in serum phospholipid concentrations between diets. S, sunflower seed oil; C, cream. Shaded area, P<O.O25.

TABLE 4. Mean percentage change in serum lipid fractions after fasting in women 3 Triglyceride Cholesterol Phospholipid 2 u- c, f3 s Diet n Day 4 Day 5 Day 4 Day 5 Day 4 Day 5 Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Sunflower seed oil, 8 fructose, starch 7-32* 7.7-37* 6.8-13* 3.3-18* 1.9-27* 2.3-30* 2.5 Cream,fructose,starch 6 +10 11.4-2 11.3 0 1.7 4-2 3.8-4 6.1-14 6.4 t, Sunflower seed oil, fructose, glucose 6-18 7.7-18* 5.1-25* 1.9-26' 2.4-23* 5.7-27* 5.6 Cream, fructose, h glucose 6 fl 7.0 0 6.0 i-3 2.4 +4 2.9-5 41-8 4.4 2 Sdower seed oil glucose, starch 7-31* 7.1-34* 5.5-18* 2.4-22* 3.1-27* 2.6-29. 4.0 H Cream,glucose,starch 7-4 64-11 48-2 2.2-2 3.0-8 3.9-9* 2.7 9 & * P<0.025. % 9

272 I. Macdonald The cholesterol concentration in subjects on fructose diets was about 10% higher than the concentration in subjects on glucose diets and this was highly significant. Similarly subjects on starch-containing diets had serum cholesterol concentrations about 10% higher than those on glucose-containing diets (Fig. 2). Phospholipid (Table 3). The concentration fell significantly in subjects on all the diets containing sunflower seed oil. Compared with the cream-containing diets, the phospholipid concentration was about 25% lower in subjects on the sunflower seed oil-containing diets. There was no difference in the effect of starch- and glucose-containing diets on phospholipid concentrations, but fructose-containing diets were associated with a lower phospholipid concentration than dietary glucose, when cream was in the diet (Fig. 3). Women There was no significant change in the weight of the subjects while they were on any of the diets. The dietary intake of each subject before the experimental diets was not evaluated. The method of expressing the change in the serum lipid fractions was the same as that used in the data for men and the mean values of the serum lipid concentration obtained during fasting before each experimental period are shown in Table 2. Triglyceride (Table 4). After 5 days on the experimental dietontaining sunflower seed oil there was a significant decrease in the concentration of triglyceride in fasting serum but no change while on the dietontaining cream. The extent of the decrease was similar to that found in the men except in the sunflower seed oil, fructose and starch diet where the decrease was significantly greater in the women. The dietontaining cream did not, after 5 days, alter the triglyceride concentration during fasting women. No significant difference was found in the response with the various dietary carbohydrates used (Fig. 1). Cholesterol (Table 4). As with the triglyceride and as found in the men, the dietontaining sunflower seed oil resulted, after 4 and 5 days, in a significant decrease in the cholesterol concentration of serum during fasting. The extent of the decrease was similar in both women and men. There was no difference in response between fructose and glucose or between starch and glucose (Fig. 2). Phospholipid (Table 4). Again, as in the other lipid fractions and as found in men, the concentration of phospholipid decreased after 4 and 5 days on dietontaining sunflower seed oil. No difference was found between the carbohydrates in causing this effect (Fig. 3). DISCUSSION The proportions of carbohydrates, fats and proteins used in these experimental diets are similar to those normally consumed and are therefore more physiological than experimental diets containing excessive proportions of one constituent, and are therefore more relevant to normal circumstances than some of the diets we have used before. Using the design of experiment reported here it is possible to uncover the influence that one dietary food group may have on another, and it is also possible to learn of small differences that may exist between one type of carbohydrate and another, differences which may be masked by the effects of other and more powerful influences. Thus the finding that in men starch results

Fats, carbohydrates and serum lipids 273 in a serum triglyceride concentration after fasting that is higher than when glucose replaces the starch, that the serum cholesterol concentration is about 10% higher after a 5 day diet with fructose than with glucose and that with cream in the diet the phospholipid concentration is greater with glucose than with thermally equivalent amounts of fructose, are differences that could be masked by the presence of other constituents of the diet. These differences were small under our conditions, but the mechanism underlying them might be of significance in long-term consumption. For example, sunflower seed oil lowers the serum cholesterol concentration, as is widely recognized, but the extent of the lowering is 10% greater when the diet contains glucose rather than fructose. There are variouontradictory reports about the effects of carbohydrates, especially sucrose, on the concentration of lipids in serum of fasting subjects (Antar & Ohlson, 1965; Dunnigan, Fife, McKiddie & Crosbie, 1970; Kuo, 1969; Macdonald & Braithwaite, 1964; Porte, Biermann & Bagdade, 1966). In view of the findings reported here, one explanation for these apparent contradictionould be that the nature of the constituents of the diet other than the carbohydrate might have masked the effect under investigation, even though that effect was present. Thus it seems that dietary fructose is associated in men, in the short term, with higher concentrations of serum triglyceride during fasting than is glucose. This difference is not immediately apparent when sunflower seed oil accompanies these two carbohydrates, but is present, even though the effect of sunflower seed oil on triglyceride concentration in serum of fasting subjects is more striking. The ability of sunflower seed oil to lower the concentration not only of cholesterol but of triglycerides and phospholipids would suggest that the effect of this polyunsaturated fat is on metabolism rather than absorption. It is possible in these experiments, however, that aream containholesterol, the decrease in serum cholesterol in the sunflower seed oil diets may be due to the cholesterol-free nature of the diet. The effect of the type of dietary fat on the concentration of lipids in serum of fasting subjects does not seem to be dependent on the sex of the consumer, whereas the lipid response to fructose does depend on the sex of the consumer, confirmation of previous findings (Macdonald, 1965). The triglyceride in the serum after a fast of at least 12 h is probably endogenous though no lipoprotein analysis was performed to coniirm the absence of chylomicrons. If exogenous lipid, ahylomicrons, had been present in any quantity, the serum triglyceride concentration after fasting would have increased and not, as was found, decreased by about 20% after 5 days on the diet containing sunflower seed oil. As endogenous serum triglyceride is mainly synthesized in the liver, using free fatty acids as the fatty acid source in the fasting state, it is difficult to understand how dietary triglyceride can interfere in this synthesis and also how the extent of synthesian be modified by the nature of the dietary fat. The sunflower seed oil must presumably either accelerate the removal of endogenous triglyceride or decrease its rate of formation, directly or indirectly. Removal of serum triglyceride is largely under the influence of lipoprotein lipase and the activity of this enzyme may be affected by the composition of exogenous lipid (Pawar & Tidwell, 1968). Perhaps the concept that endogenous triglycerides are mainly influenced by dietary carbohydrate has arisen from studies in which the diet haontained little polyunsaturated fat. It seems that, in dietontaining relatively little polyunsaturated fat, carbohydrate is the major influence affecting the endogenous triglyceride concentration after a 12-14 h fast, but this

274 I. Macdonald effect of carbohydrate can, it seems, be masked by the action of sunflower seed oil. The fasting triglyceride concentration may possibly be determined by the ratio: amount and type of dietary carbohydrate/amount and type of dietary fat. ACKNOWLEDGMENTS I am very grateful to the volunteers, and to Beecham Products for a grant. REFERENCES AHRENS, E.H., HIRSCH, S., LITTLE, K., FARQUHAR, J.W. & STEIN, Y. (1961) Carbohydrate-induced and fatinduced lipaemia. Transactions of the Association of American Physicians, 74, 134-146. ANTAR, M.A., LITTLE, J.A., LUCAS, C., BUCKLEY, G.C. & CSIMA, A. (1970) Interrelationship between the kinds of dietary carbohydrate and fat in hyperlipoproteinaemic patients. Atherosclerosis, 11, 191-201. ANTAR, M.A. & OHLSON, M.A. (1965) Effects of simple and complex carbodydrates upon total lipids, nonphospholipids, and different fractions of phospholipids of serum in young men and women. Journal of Nutrition, 85, 329-331. BLOCK, W.D., JARRETI-, K.J. & LEVINE, J.B. (1966) An improved automated determination of serum cholesterol with a single colour reagent. Clinical Chemistry, 12, 681489. DUNNIGAN, M.G., FIFE, T., MC~DIE, M.T. & CROSBIE, S.M. (1970) The effects of isocaloric exchange of dietary starch and sucrose on glucose tolerance, plasma insulin and serum lipids in man. Clinical Science, 38, 1-9. KEYS, A. (1970) Coronary heart disease in seven countries. Circulation, 41, Supplement 1, 1-211. KREHL, W.A., LOPEZ, A. &GOOD, E.I. (1967) A rapid analytical system for determining serum lipids. American Journal of Clinical Nutrition, 20,968-917. Kuo, P.T. (1969) Metabolic basis of human atherosclerosis. Metabolism, 18, 631-634. LOFLAND, H.B., JR (1964) A semi-automated procedure for the determination of triglycerides in serum. Analytical Biochemistry, 9, 393-400. MACDONALD, I. (1965) The lipid response of young women to dietary carbohydrates. American Journal of Clinical Nutrition, 16, 458463. MACDONALD, I. (1967) Interrelationships between the influences of dietary carbohydrates and fats on fasting serum lipids. American Journal of Clinical Nutrition, 20, 345-351. MACDONALD, I. & BRAITHWAITE, D.M. (1964) The influence of dietary carbohydrates on the lipid pattern in serum and in adipose tissue. Clinical Science, 27,23-30. MCCANCE, R.A. & WIDDOWSON, E.M. (1960) The Composition of Foods. M.R.C. Special Report No. 235, 3rd edn. PAWAR, S.S. & TIDWELL, H.C. (1968) Effect of ingestion of unsaturated fat on lipolytic activity of rat tissues. Journal of Lipid Research, 9, 334-336. PORTE, D., JR, BIERMANN, E.L. & BAGDADE, J.D. (1966) Substitution of dietary starch for dextrose in hyperlipaemic subjects. Proceedings of the Society for Experimental Biology and Medicine, 123, 814-816.